Identification of treatment‐induced vulnerabilities in pancreatic cancer patients using functional model systems

Abstract Despite the advance and success of precision oncology in gastrointestinal cancers, the frequency of molecular‐informed therapy decisions in pancreatic ductal adenocarcinoma (PDAC) is currently neglectable. We present a longitudinal precision oncology platform based on functional model systems, including patient‐derived organoids, to identify chemotherapy‐induced vulnerabilities. We demonstrate that treatment‐induced tumor cell plasticity in vivo distinctly changes responsiveness to targeted therapies, without the presence of a selectable genetic marker, indicating that tumor cell plasticity can be functionalized. By adding a mechanistic layer to precision oncology, adaptive processes of tumors under therapy can be exploited, particularly in highly plastic tumors, such as pancreatic cancer.

Dear Dr. Durdevic, Thank you for considering our manuscript. Certainly, we respect your decision and it is not our common way to rebuttal a decision. However, in this particular case, we both agreed to kindly ask you to reconsider your decision, since it comes as a major suprise to us. We both presented this interdisciplinary work in several talks and traced always an amazing feedback across the fields -even in the zoom age we are living in. As a reviewer EMBO Molecular Medicine for Lise Roth, we are convinced that the manuscript is a very good match for the Journal.
With this work, we are the first group to show how treatment-imposed pressure can be exploited therapeutically in pancreatic cancer and we provide the exact platform for such an approach. For pancreatic cancer, we have clear evidence that precision oncology is a substantial benefit for patients but only 1 out of 4 patients has an actionable genetic lesion. We are the first group clearly demonstrating that functionalizing human organoid models expands the group of patients with a rational therapeutic option. This is an important conceptual advance we are transmitting by our work.
Furthermore, our work gives completely new insights into cellular plasticity in the clinical course of the disease and, therefore, is of high translational significance as well as of broad interest to the field of molecular medicine and personalized oncology. In addition, our work represents a major conceptional advance as we clearly show that tumor de-differentiation is not a default adaptive mechanism in tumor cells being exposed to chemotherapy but rather one of many. Specifically, we demonstrate on a morphologic and molecular level that tumor cells after chemotherapy in vivo are reprogrammed to be more epithelial. Importantly, this reprogramming opens new avenues for targeted therapies. Of note, this reprogramming is not driven by clonal selection and an altered mutational profile. Therefore, we propose to implement patient-derived functional model systems which are able to recapitulate adaptive processes under chemotherapy into the clinical workflow, especially, in highly plastic cancers. Thank you for the submission of your manuscript to EMBO Molecular Medicine. We have now received feedback from the three reviewers who agreed to evaluate your manuscript. As you will see from the reports below, the referees acknowledge the interest of the study but also raise important critique that should be addressed in a major revision.
Further consideration of a revision that addresses reviewers' concerns in full will entail a second round of review. EMBO Molecular Medicine encourages a single round of revision only and therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript. For this reason, and to save you from any frustrations in the end, I would strongly advise against returning an incomplete revision.
We would welcome the submission of a revised version within three months for further consideration. However, we realize that the current situation is exceptional on the account of the COVID-19/SARS-CoV-2 pandemic. Please let us know if you require longer to complete the revision.
I look forward to seeing a revised form of your manuscript as soon as possible. Use this link to login to the manuscript system and submit your revision: https://embomolmed.msubmit.net/cgi-bin/main.plex 1. The authors should demonstrate the targeting efficacy of the MEK inhibitors in the organoid cultures and cell lines experiments. A western blot or IHC/IF analysis of pERK will suffice to address this comment. 2. The FOLFIRINOX in vitro formulation should be clarified. Are 5FU and Irinotecan given simultaneously? 3. The Cell-Titer experiments should be validated by a second method that does not utilize a metabolic marker to measure cell viability. 4. The experiments lack details on the passage of the organoid cultures for each of the results. A copy number profile should be done for each of the passage to make sure that the organoid is genetically stable and remain representative of the primary tumor. It will preferable if the copy number analysis is accompanied by marker analysis using IHC/IF. 5. Most important, these initial findings in two cases should be extended to additional ones to define usefulness of the platform in the clinical setting.
Referee #2 (Comments on Novelty/Model System for Author): the personalized treatment is necesary for patients with PDAC. This paper describes one realistic possibilty.
Referee #2 (Remarks for Author): In this paper Katja Peschke and colleagues describes a longitudinal precision oncology platform, which point to the value of mechanistic investigation to advance concepts for PDAC targeting, and report a case to illustrate its efficiency. This is an original manuscript which describes a promising approach to improve the PDAC treatment particularly by applying drugs which are not still employed in clinics. This type of platform will help in the future to the clinicians to select the better therapeutic strategy for each patient. I suggest some points to improve this paper before it is accepted.

Comments
This paper demonstrate that changes in sensitivity to drugs were not associated to evident changes in their genetic landscape which, in my opinion, strong suggest that resistance to the treatments is associated to the phenotype of the cells rather they genotype modifications, at least in PDAC. This point merit to be discussed more in details in the manuscript. Another important point to be discussed is about the place of the molecular markers which should be associated to some chemo-sensitivities ? Are all the PDACs providers of cells or organoids for applying systematically this approach ? Is the response of each model concordant ? Why select organoids rather primary cell cultures or vice versa ? In practical terms, what is the necessary time to obtain conclusive results from a give patient? This is determinant for its clinical utilization given the short expectancy of survival of these patients, particularly for non-operable patients.
The strength of the manuscript is the longitudinal analysis of treatment-naive samples and after neoadjuvant therapy in pancreatic ductal adenocarcinoma. Similar approaches have been considered but to the best of my knowledge, there are no longitudinal studies reported. The authors present a pilot study with an integrative analysis of genetics, molecular and pharmacotyping of patient-derived organoids that could be implemented in the management of pancreatic ductal adenocarcinoma.
Referee #3 (Remarks for Author): The manuscript by Pescheke K, Jakubowsky H, et al., entitled "Identification of treatment-induced vulnerabilities in pancreatic cancer patients using functional model systems" studies the molecular and genetic evolution of tumor cells after neoadjuvant therapy from one patient of pancreatic ductal adenocarcinoma (PDAC). The authors generated tumor-derived organoids and cell lines to investigate genetic and phenotypic adaptations to treatment, and differential drug sensitivity to identify treatment-induced vulnerabilities. They concluded that FOLFIRINOX neoadjuvant treatment induces resistance to FOLFIRINOX and sensitivity to MEK inhibitors independent of a genetic marker. The strength of the manuscript is the longitudinal analysis of tumor samples.
Although it is a case study about one patient, which limits the impact in the general population of PDAC patients, the study is timely and relevant as it serves as proof of concept for developing strategies to inform clinical decisions in the management of PDAC. Major concerns: The authors claim that therapy induces phenotypic adaptations of tumor cells; however, the data do not exclude pre-existing clones with different sensitivity to FOLFIRINOX in the treatment-naïve sample generated by non-mutational mechanisms. In my opinion, the focus on the phenotypic switch induced by therapy is an oversimplification, and non-mutational mechanisms of tumor heterogeneity should be considered in the interpretation and discussion of the data. The longitudinal study deserves additional characterization in the diagnostic specimens (pretreatment and resection) and functional model systems. Additional qualitative and quantitative experiments should focus on elucidating epithelial and stromal heterogeneity in naïve and neoadjuvant samples by for example immunofluorescent analysis (e.g., markers of molecular subtypes, fibroblast, and immune infiltration, etc) I am not convinced that SLUG expression in neoadjuvant treatment explains the acquired sensitivity to MEK inhibitors upon FOLFIRINOX treatment. Additional molecular analysis (e.g., gain and loss of function in patient-derived organoids, etc) should be performed if the authors wish to include this analysis. Lastly, the authors report a longitudinal precision oncology platform, which could be in principle tested in prospective clinical trials. To strengthen the manuscript's impact and help future studies, the authors could consider including a description of the manpower, technical and funding resources, and time required to implement such a platform in the clinical management of PDAC. Such a description should highlight the limitations as well. Minor concerns: • Figure 1 is overcrowded.
• "In an interim staging by 18F-FDG PET-MRI, the glucose metabolism (as measured by the standard uptake value) was markedly reduced while the tumor size was unaltered, indicating a metabolic switch (Fig. 1C)" Can PET scanning be considered quantitative? Would it be possible that FFX is selectively killing highly cycling cells, which presumably also consume more glucose? • Provide the nomenclature of the staging system • "Histologically, both biopsies demonstrated a well to moderate differentiation (Fig. 1D)." The histological analysis deserves a high resolution image and . Also, is the magnification of ID188 and ID211 the same? • PDOs isolated pre-chemotherapy (ID188) showed a lumen filling growth pattern and revealed a quasi-mesenchymal growth in 2D (Fig. 1E). PDOs isolated from the resection (ID211) were transparent and grew as an epithelial monolayer with colony forming growth in 2D (Fig. 1E) Provide a reference were the growth patter of PDOs is associated with molecular subtypes or differentiation.

The FOLFIRINOX in vitro formulation should be clarified. Are 5FU
and Irinotecan given simultaneously?
The detailed formulation and treatment sequence has been added to the M&M section. We apologize for the brevity previously. Specifically, the following paragraph has been added: For in vitro FOLFIRINOX treatment, a mixture of 5-Fluoruracil (c max = 37.6 µM), Irinotecan (cmax = 16.9 µM) and Oxaliplatin (cmax = 7.9 µM) was prepared according to the ratio in clinical practice and added simultaneously in a 7-point drug dilution for 72 hours.

The Cell-Titer experiments should be validated by a second method that does not utilize a metabolic marker to measure cell viability.
We appreciate this insightful comment. We now have quantified  The reviewer raises an important issue here. Now, we have indicated the specific passages of primary cells or organoids in each experiment. In addition, we have previously performed whole exome sequencing in early passage PDOs (passages P5-10) and at late passage (above passage 50). We confirmed that the genomic profile of PDOs is fairly stable across passages as exemplified in PDO ID25 below. We still believe that longterm culture and passaging alters organoid biology. Therefore, as QC measure in our translational workflow involving PDOs, we have included in our SOP that all functional assays have to be performed between passage 5 and 30. We have indicated the passage number of each experiment in the revised version of the manuscript. We agree with the reviewer that our platform and translational implementation of organoid technology in general, needs to pass test of time by validation in co-clinical trials. At the same time, here, we present a n-of-1 study to underscore the value of these type of patient-derived model systems facilitating personalized oncology particularly in pancreatic cancer where pure genome-driven approaches frequently fail to identify targeted therapies. To be precise, in the "know your tumor" clinical PDAC trial, in 75% of patient with a complete molecular diagnostic work-up, no targetable lesion was observed. Therefore, we are convinced, with improvements in culture and screening technologies which are currently under development, the functional and mechanistic approach described in the manuscript will fill an important gap.
Importantly, the plastic tumor cells behavior in response to chemotherapeutic therapy is not a universal mechanism of resistance or tumor evolution. We know that in certain instances, e.g., clonal selection is a main driver of adaption to treatment. Nevertheless, plasticity is an important mechanism in this context which has been postulated for years and heavily investigated by the basic science community, however, never really shown in a clinical longitudinal setting using patient-derived models. We believe that this is one important strength of our study and we therefore have implemented longitudinal sampling in our standard workflow to expand upon these findings in the near future. In addition, we kindly ask the reviewer to consider data already in the manuscript, which underscore the usefulness of the approach. When we compared the sensitivity of ID188 and ID211 to MEKi to a large panel of patient-derived cell lines, we observed that ID188 is one of the most resistant and ID211 one of the most sensitive lines. To underscore and visualize this finding, we have included an illustration of these important finding as new Fig.   2K.

Reviewer 2
2.1. This paper demonstrates that changes in sensitivity to drugs were not associated to evident changes in their genetic landscape which, in my opinion, strong suggest that resistance to the treatments is associated to the phenotype of the cells rather they genotype modifications, at least in PDAC. This point merit to be discussed more in details in the manuscript.
We thank the reviewer for time, effort and constructive comments. We agree that the description that non-genetic mechanisms contribute to changes in drug sensitivity induced in humans in vivo, is a major strength of the work. This will open new research directions, since such data underscore the need to systematically address the molecular underpinning. We increased the discussion and cite a current review article describing the complexity of non-genetic resistance mechanisms.

Another important point to be discussed is about the place of the molecular markers which should be associated to some chemosensitivities?
We appreciate the comment and elaborated further on molecular markers and therapy in the discussion section. Specifically, the following sentences have been added: "As indicated above, real-world outcomes suggest that genetic profiling followed by molecularly tailored therapy is The reviewer makes a key point here. We have included longitudinal sampling to our routine workflow recently. In addition, we have put additional IRB-approved protocols into place allowing longitudinal sampling not just in a neoadjuvant setting but also at a metastatic stage to explore this important aspect of PDAC biology more systematically in the future.
The main reason for generating 2D lines from PDOs is that our roboticaugmented screening platform allows testing of our 415-drug library of adherent cell in 11 days. Doing this screen in 3D would currently take triple the amount of time and costs. We are working on pipelines to automatize also the 3D screen and reduce the costs for PDO screening.
However, so far, we have subsequently validated all hits of the screen in 2D also in 3D organoid culture with extremely high concordance. Having this said, this approach does not exclude false negative results of the high-throughput screen and therefore, robustly validating hits, as exemplified for the MEKi, is necessary. We have added this information in text as well as the supplemental methods. We thank the reviewer for the important insights and the efforts to improve our manuscript. We completely agree with the reviewer. We believe non-mutational or epigenetic mechanisms whether they are preexisting or acquired are so far under-investigated areas of PDAC biology.
Our RNAseq results clearly indicate significant changes in transcriptional programs upon treatment. Some of these altered pathways such as TGFbeta signaling have pronounced impact upon morphology and phenotype. We did not intend to oversimplify and reduce the impact of epigenetic mechanisms; however, the phenotype was one of the first findings that jumped to the eye when comparing these PDO lines. In response to the reviewer, we included a paragraph clearly stating that the development of resistance involves multiple processes including evolution under selection pressure of a therapeutic intervention.
However, we cite also a recent review article that non-genetic events are currently emerging as contributors of therapy resistance. In sum, we believe that evidence provided by our work will stimulate systematic research into non-genetic events of therapy resistance in PDAC, needed to comprehensively understand all facets of a major clinical problem.

The longitudinal study deserves additional characterization in the diagnostic specimens (pretreatment and resection) and functional model systems. Additional qualitative and quantitative experiments should
focus on elucidating epithelial and stromal heterogeneity in naïve and neoadjuvant samples by for example immunofluorescent analysis (e.g.,

markers of molecular subtypes, fibroblast, and immune infiltration, etc)
We thank the reviewer for this comment. Unfortunately, there was no treatment-naïve tissue left as the FNB tissue was used for diagnostics and H&E. We agree that it would have been interesting to study changes in the composition of the tumor microenvironment. Yet again, it is not clear how representative these changes would have been for other patients. To further characterize our patient-derived models we have added additional data on differences in proliferation, GLUT1 expression and expression of EMT markers (see Figure EV1B).

I am not convinced that SLUG expression in neoadjuvant treatment explains the acquired sensitivity to MEK inhibitors upon FOLFIRINOX treatment. Additional molecular analysis (e.g., gain and loss of function in patient-derived organoids, etc) should be performed if the authors wish to include this analysis.
We agree with the reviewer that the acquired sensitivity towards MEKi is likely caused by multifactorial effects of chemotherapy. SLUG expression has been indicated in this context but as indicated by our gene expression analyses it is way more complex than just EMT reprogramming. Therefore, we decided to move this figure to the supplement and we tune down the statement by including the note that other mechanisms might contribute.

Lastly, the authors report a longitudinal precision oncology
platform, which could be in principle tested in prospective clinical trials. Here, the reviewer makes an excellent point regarding feasibility of the platform in a co-clinical trial setting. We have added additional information in the revised manuscript (Fig.EV2G). This illustration places the time frame of our longitudinal translational platform in context of the median survival of standard-of-care therapies. As already stated, we are convinced that for selected patients, such a longitudinal platform will allow mechanistic data (e.g. drug screening) into clinical decision making.
In addition, there several strategies to streamline the workflow and increase feasibility as also indicated in response to reviewer 2 (see 2.4).

Experiment
Cell number

Figure 1 is overcrowded.
We thank the reviewer for pointing this out. We are restricted by the Report format to two figures. However, we re-arranged figure 1 to increase clarity. Therefore, we consider PET scanning as quantitative.

"In an interim staging by 18F-FDG PET-MRI
We cannot exclude that FFX is selectively killing highly cycling cells, but we include data in the revised version, demonstrating that the post-CTX 3D model showed increased proliferation (Fig. EV1C, EV2C/D Supplementary Figure 1C and 2C/D). This data argues against a selective killing of cancer cells with high proliferative capacity. Since we cannot exclude differential influence of the culture medium, we do not discuss the possibility.
Taken together, although we cannot revisit and asses the primary tissue for proliferation as none of the FNB tissue is available anymore, our organoids indicate that proliferation is not confounding our conclusions.

Provide the nomenclature of the staging system
The patient was staged according the TNM Classification Edition 8, 2017.

"
Histologically, both biopsies demonstrated a well to moderate differentiation (Fig. 1D)." The histological analysis deserves a highresolution image and, also, is the magnification of ID188 and ID211 the same?
We have adjusted the magnification und increased resolution (please see Figure 1).

PDOs isolated pre-chemotherapy (ID188) showed a lumen filling
growth pattern and revealed a quasi-mesenchymal growth in 2D (Fig.   1E). PDOs isolated from the resection (ID211) were transparent and grew as an epithelial monolayer with colony forming growth in 2D (Fig.   1E). Provide a reference were the growth pattern of PDOs is associated with molecular subtypes or differentiation.
We thank the reviewer for this important comment. Indeed, in a recent collaboration, the team demonstrated that oncogenic transformation by Kras G12D with or without loss of CDKN2A is accompanied by EMT and lumen-filling phenotype in a human pluripotent stem cell-derived

Subtyping in Pancreatic Cancer"
We apologize for this negligence and provide the appropriate citation in the revised version of the manuscript.
3.11. Fig 1F. Validation of the results by immunofluorescence with markers of classical and basal-like (e.g.; GATA6 and KRT5 respectively) In concordance with the switch from a lumen-filling phenotype to a spheric, cystic organoid growth pattern, we observed a mesenchymal-toepithelial-transition (MET) protein expression pattern by western blot indicated by decreased KRT81, increased E-cadherin and GATA6 abundance in the ID211 2D culture (Fig. EV1D). This is concordant with the changes in the mRNA expression of SNAI2 and Vimentin in the 3D models (see Fig. EV1E). These results underscore our overall conclusion of a re-differentiation occurring upon FFX in vivo. Epiphenomenon in this context refers to the limitation just using PDOs derived from one single patient. How can we be sure that the chemotherapy-induced vulnerability towards MEKi is meaningful or just a secondary, unrelated phenomenon (epiphenomenon) or coincidence?

Importantly
To avoid misunderstandings, we have rephrased the sentence and added an illustration for clarification (Fig. EV2K).

SLUG. The correct gene symbol is SNAI2
We thank the reviewer for pointing out this mistake which has been corrected in the revised version of our manuscript. Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: 1) Please address all the points raised by the referee #1. 2) In the main manuscript file, please do the following: -Move supplementary methods to main manuscript file.
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Referee #3 (Comments on Novelty/Model System for Author): The manuscript will be relevant for the audience of EMBO molecular medicine Referee #3 (Remarks for Author): The authors have addressed all my concerns We thank the reviewer for the helpful comments. We added the sentence "The events directing plasticity are often mediated by epigenetic regulation and chromatin remodeling and their understanding is of great value to establish plasticity blocking therapies.", to underscore the value of plasticity in adaption and

resistance.
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